A prime example for neurological degenerations is represented by Alzheimer's disease (AD). AD is a devastating dementia affecting approximately 4 million people in Europe. It progressively destroys a person's memory, ability to learn, to reason, make judgments, communicate, and carry out daily activities. Up to date, there is no effective treatment or cure for Alzheimer's disease. On average, a person dies 8 years after the first symptoms arise. Patients are commonly treated with acetylcholinesterase inhibitors and/or NMDA-receptor antagonists. These drugs show modest clinical benefit in mild to moderate cases of Alzheimer. They are only efficient for up to 12 months, as their beneficial effects fade. Nevertheless, the market value for these drugs amounted to $ 4 billion in 2005 and is expected to rise to approximately $ 6 billions by 2010. More importantly, the National Institute on Aging estimated that medical care costs for the 4.5 million Alzheimer patients in USA amounted to $ 100 billion annually, rising to $160 billions by 2010. As the number of Alzheimer patients is expected to quadruplicate within the next 40 years, the increase in care costs may exceed the ability of health systems to absorb these costs. Therefore, the efficient and fast development of Alzheimer drugs is not only eagerly anticipated by patients and the pharmaceutical industry, but it is a necessity for all industrialized countries. The Alzheimer association estimated that a treatment that would efficiently delay the onset of AD for 5 years could save $ 50 billion annually in the US.
Given the complex pathological mechanisms, drug development programs for neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, Multiple Sclerosis (MS), bovine spongiform encephalopathy (BSE), Creutzfeldt Jacob disease (CJD), and various retinal degenerations predominantly depend mainly on whole animal models which are very expensive, laborious, and time consuming. Analyses of drug effects or pathological mechanisms are predominately performed by cell-destructive procedures like immunocytochemical, molecular biological, and/or proteinchemical methods.
But also non-destructive and labeling free measuring principles like impedance spectroscopy are known. Changes in impedance can be caused by alterations of intracellular or extracellular processes that have been induced by e.g. transformation of non-pathological into pathological form. Although a large number of transgenic animal models, ex vivo cultures, or cell lines for neurodegenerative diseases have been established, up to now, impedance spectroscopy has not been used for the analysis of organotypic tissues e.g. tissues related to neuronal degenerations. In most instances impedance-based screening has been carried out on monolayer cultures, which have the disadvantage that they do not take into account the three-dimensional geometry of the in vivo situation.
Since impedance spectroscopy is a non-invasive method, long-term measurements can be realized without influencing cellular behaviour. Hence, the cellular read out reflects real time conditions without disturbing effects due to complex and long lasting physical procedures. The latter methods are well suited to study a broad range of biological and medical problems, however, in many cases the real cellular information dropped away since e.g. staining artifacts make it difficult or completely impossible to interpret the extracted cellular data. In principle, tracing of biological processes in living cells can be performed with modern labeling techniques, but hold the risk to falsify data due to the positioning of foreign substance within the cell itself.
Impedance spectroscopy—also known as cellular dielectric spectroscopy (CDS) or electric impedance spectroscopy (EIS)—can be used to measure frequency dependent alterations of passive electrical properties of single cells by applying defined alternate currents and/or voltages.
The bio-impedance of single cells can be measured with a working electrode and a counter electrode. Different cellular parameters such as the capacitance and resistance of the cell membranes as well as intracellular membranes of organelles, the resistance of the extracellular medium and intrinsic cytoplasm, the extracellular matrix and the contact between cell and electrode contribute to the overall cellular impedance. To analyze alterations of impedance of living cells, an alternate voltage is applied to a biological sample. Depending on the dielectric properties of sub-cellular compartments and molecules the applied current can flow from an active working electrode through the cells whereby the remaining current is collected by a counter electrode. Depending on the frequency of the applied voltage, alterations of certain cellular compartments can be identified.
In this context it is possible to discriminate cellular behaviour according to their dispersions which can be divided in α-, and γ-dispersion. The α-dispersion ranges from 1 Hz to 1 kHz and results from counter ions, glycocalyx, and from ion channels, whereas the β-dispersion (1 kHz-100 MHz) is due to the cytoplasm membrane, intracellular membranes (organelles), cytosol and proteins. Additionally, the γ-dispersion (100 MHz-100 GHz) is defined by the dielectric properties of free and bound water, relaxation of charged subgroups, and partially by protein-protein interactions. In particular, the frequency dependent measurement of manifold cellular alterations of both electrical and non-electrical active cells under non-destructive real-time conditions point out the infinite possibilities of this technique.
A commonly used impedance recording method is the so-called electric cell-substrate impedance sensing (ECIS) introduced by Giaever and Keese (Giaever, I., and Keese, C. R. Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci USA. 1984; 81(12):3761-3764; U.S. Pat. No. 5,178,096). For ECIS, cells have to grow on a small gold electrode implemented on the bottom of a culture dish. If an alternate voltage is applied between a small working electrode, attached cells, and a large counter electrode, an increased impedance can be observed at a given time and single frequencies. ECIS has been further optimized for automated, non-invasive, real-time, and high throughput analysis (WO 2007/015878; WO 2006/104839; WO 2005/077104; WO2004/010103; Wegener et al. Impedance analysis of epithelial and endothelial cell monolayers cultured on gold surfaces. J Biochem Biophys Methods. 1996; 32(3):151-170; Ciambrone et al. S. Cellular dielectric spectroscopy: a powerful new approach to label-free cellular analysis. J Biomol Screen. 2004; 9(6):467-480). These impedance-based multi-well devices have been used for recording of healthy, non-pathological adherent cells (monolayer cultures) for detecting cell attachment, detachment, migration, cell-substrate interaction, blood-brain-barrier function, chemotaxis, toxicology, proliferation, ligand-receptor-interaction, and apoptosis after application of test substances. In each of these cases cells were cultured as monolayer and analyzed by impedance spectroscopy. However, measuring the impedance in monolayer cultures does not provide data referring to the three-dimensional structure of living tissue.
There are also several approaches using impedance-based sensors for use in living systems. Heroux and Bourdages have published an article entitled “Monitoring living tissues by electrical impedance spectroscopy” (Ann Biomed Eng. 1994 May-June; 22(3):328-37). The article refers to the development of an electrical impedance spectroscopy (EIS) probe for monitoring cellular changes in living animals. This probe comprises two slender (0.17 mm) electrodes connected to two miniature coaxial cables and fixed at a distance of 5 mm from each other using an insulating plate. For impedimetric analysis the probe was directly implanted in living animals for monitoring different tissues (brain cortex, liver, kidney, spleen, and muscle). Impedance recording was performed after pentobarbital-induced respiratory and cardiac arrest. However, the above described assays using living animals are not suitable for long term impedance measurements and do not provide an automated screening method for drug candidates for the treatment of specific diseases.
WO 2006/047299 discloses an organotypic slice assay that can be used to study neurodegenerative diseases. The invention includes the generation of brain slice cultures and their possible analysis by HPLC, ELISA, MALDI, SELEX, gene arrays, or immunochemical assays. The invention also describes electrophysiological recordings of action potentials by means of whole-cell voltage- and current clamp technique. However, WO 2006/047299 does not disclose a method or device for measuring impedance in organotypic tissue.
Since impedance-based recordings of organotypic cultures have not been possible so far, a device and method for simple, fast, cost effective, non-destructive, and free-free measurement of cellular parameters of pathological and non-pathological tissues especially obtained from different parts of the central nervous system of individual (animal or human being) is desired.
The methods and devices for measuring impedance in organotypic tissue as defined in the claims overcome at least some of the problems of the prior art.